Stabilization of whole blood at room temperature

ABSTRACT

The present disclosure is directed to the stabilization of nucleated cells in a whole blood sample ex-vivo, effected by an additive being a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, preferably a mitochondria-targeted antioxidant selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof. In a specific embodiment, the liquid composition further comprises a protease inhibitor. Further provided is advantageous use of the composition for stabilizing an analyte selected from DNA, RNA and protein in intact nucleated cells of the whole-blood sample ex-vivo, as well as kits including the composition for practicing said use. Additionally, specific methods are provided for stabilizing intact nucleated cells of a whole-blood sample ex-vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/EP2015/051658 filed Jan. 28, 2015, and claims priority toEuropean Patent Application No. 14153199.6 filed Jan. 30, 2014, thedisclosures of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE DISCLOSURE

The present disclosure pertains to compositions, uses, kits and methodsuseful in the stabilization of intact viable cells, particularlynucleated cells present in whole blood samples. The present disclosureis directed to the stabilization of nucleated cells in a whole bloodsample ex-vivo, effected by an additive being a liquid composition forstabilizing an analyte in intact nucleated cells of a whole-blood sampleex-vivo, the composition being an aqueous solution, the solutioncomprising an anticoagulant, a phosphate salt, a cell-metabolizablesugar, adenine, and an antioxidant, wherein the antioxidant comprises amitochondria-targeted antioxidant, preferably a mitochondria-targetedantioxidant selected from the group consisting of SkQ1, MitoQ, SS-31,and a mixture thereof. In a specific embodiment, the liquid compositionfurther comprises a protease inhibitor. Further provided is advantageoususe of the composition for stabilizing an analyte selected from DNA, RNAand protein in intact nucleated cells of the whole-blood sample ex-vivo,as well as kits including the composition for practicing said use.Additionally, specific methods are provided for stabilizing intactnucleated cells of a whole-blood sample ex-vivo.

Whole blood is one of the most frequently collected sample material fordiagnostic analysis in medicine. However, sampling and analysis of wholeblood are usually separate events. Following the sampling step, a majortechnical challenge exists concerning stabilization of intact cells,particularly intact nucleated cells, over a period of time. There is aspecific desire in the art to preserve the biological status ofnucleated cells in a whole blood sample, in order to bridge the timeinterval between sampling and analysis, see Olson W C et al. (Journal ofTranslational Medicine 9 (2011) 26), Tanner et al. (Clin. Lab. Haem. 24(2002) 337-341), Baechler et al. (Genes and Immunity 5 (2004) 347-353),and Elliott et al. (International Journal of Epidemiology 37 (2008)234-244). “Preservation of biological status” or “stabilization” isunderstood as providing conditions under which changes concerning thecomposition and levels of biomolecules present in a living cell at thetime of sampling are minimized while keeping the cell intact and viable.Particular biomolecules of interest in this regard are DNA, RNA andprotein.

Although some cell-preserving additives are known to the art, it stillremains a technical challenge to stabilize a whole blood sample withnucleated cells over a period of 2 to 3 days at room temperature. Thereis a particular need for means enabling isolation of intact nucleatedcells after that time interval, wherein the cells are preferably viable.More specifically, it is desired that any change of composition andlevel of biomolecules in the cells is minimized such that analysis ofthe cells after the time interval closely reflects the biological statusof the cells at the time point when the whole blood was sampled.

BRIEF SUMMARY OF THE DISCLOSURE

A first aspect reported herein is a liquid composition for stabilizingan analyte in intact nucleated cells of a whole-blood sample ex-vivo,the composition being an aqueous solution, the solution comprising ananticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine,and an antioxidant, wherein the antioxidant comprises amitochondria-targeted antioxidant, preferably a mitochondria-targetedantioxidant selected from the group consisting of SkQ1, MitoQ, SS-31,and a mixture thereof.

A second aspect reported herein is the use of a composition as disclosedherein for stabilizing an analyte selected from DNA, RNA and protein inintact nucleated cells of a whole-blood sample ex-vivo.

A third aspect reported herein is a kit of parts comprising a blooddrawing container enclosing a liquid composition according to thedisclosure herein, the kit further comprising packaging material, alabel, and a user instruction sheet.

A fourth aspect reported herein is method for stabilizing intactnucleated cells of a whole-blood sample ex-vivo, the method comprisingthe steps of (a) providing the whole blood sample ex-vivo; (b)contacting and mixing the sample of step (a) with a compositionaccording to the disclosure herein; (c) incubating the mixture obtainedin step (b), thereby stabilizing intact nucleated cells of the ex-vivowhole-blood sample.

DESCRIPTION OF THE FIGURES

FIG. 1 Detection of MDA-MB-468 cells spiked into blood samples afterstorage for 48 hours at room temperature. Depicted is the percentage ofMDA-MB-468 cells detected by immunostaining with Anti-CK5/8 antigens.“Box-and-whisker plot” indicating mean percentage of target cellretrieval by way of assay as described in Example 1. 1st diagrammedbox—EDTA: MDA-MB-468 cells detected in blood sample stored in EDTA asanticoagulant. 2nd box—EDTA+Sivelestat: MDA-MB-468 cells detected inblood sample stored in EDTA as anticoagulant and sivelestat asstabilizer. 3rd box—EDTA+Pefabloc® SC: MDA-MB-468 cells detected inblood sample stored in EDTA as anticoagulant and Pefabloc® SC asstabilizer. 4th box-EDTA+SKQ1: MDA-MB-468 cells detected in blood samplestored in EDTA as anticoagulant and SKQ1 as stabilizer. 5thbox—EDTA+MitoQ: MDA-MB-468 cells detected in blood sample stored in EDTAas anticoagulant and MitoQ as stabilizer. 6th box—EDTA+glutathione:MDA-MB-468 cells detected in blood sample stored in EDTA asanticoagulant and glutathione as stabilizer.

FIG. 2 Detection of MDA-MB-468 cells spiked into blood samples afterstorage for 48 hours at room temperature. Depicted is the percentage ofMDA-MB-468 cells detected by immunodetection with fluorescent anti-CK5/8 antibodies. “Box plot” indicating mean percentage of target cellretrieval by way of the assay described in Example 2. 1st diagrammedbox—EDTA: MDA-MB-468 cells detected in blood sample stored in EDTA asanticoagulant. 2nd box—CPDA: MDA-MB-468 cells detected in blood samplestored in citrate/citric acid, phosphate, dextrose and adenine. 3rdbox—CPDA+SS-31: MDA-MB-468 cells detected in blood sample stored incitrate, phosphate, dextrose, adenine and SS+31. 4thbox—CPDA+glutathione: MDA-MB-468 cells detected in blood sample storedin citrate, phosphate, dextrose, adenine and glutathione.

FIG. 3 Influence of storage on DNA yield. Samples were stored in EDTA orEDTA supplemented with glutathione, SkQ1 and Pefabloc® SC (“Stabilizer3”), as indicated in Example 3.

FIG. 4 Blood from two blood donors was collected either in EDTA or instabilizer as described in Example 5. Samples were spiked withMDA-MB-468 cells. Immediately and following a 72 hours long incubationat room temperature, the MDA-MB-468 cells were isolated and the nucleicacids were extracted. The set of nucleic acid parameters depicted in thefigure was amplified by RT-PCR or PCR (depending on the template);changes of the respective nucleic acid levels were calculated using GCKDNA as endogenous reference. “Stabilizer 72 h” refers to the samplesincubated for 72 hours with Stabilizer 5 as described in Example 5.

FIG. 5 Blood from 2 different donors was either stabilized with EDTA orwith stabilizer. MDA-MB-468 cells were spiked to the blood samples. Thesamples were stored for 48 hours at room temperature. MDA-MB-468 cellswere enriched by erythrocyte lysis and immunomagnetic isolation usingCD326 (EpCAM) Microparticles (see Example 7). The cells were culturedfor more than 100 hrs.

Open diamond: specimen 1, EDTA-stabilized, closed diamond: specimen 1with Stabilizer 5.

Open square: specimen 2, EDTA-stabilized, closed square: specimen 2 withStabilizer 5.

Open circle: specimen 1, EDTA-stabilized, closed circle: specimen 1 withStabilizer 5.

Open triangle: specimen 2, EDTA-stabilized, closed triangle: specimen 2with Stabilizer 5.

DETAILED DESCRIPTION

For the purpose of the present disclosure, certain terms are defined asfollows herein. In the event of a conflict in a definition in thepresent disclosure and that of a cited reference, the present disclosurecontrols.

As used herein, the term “comprising” means that other steps and othercomponents that do not affect the end result may be utilized. The term“comprising” encompasses the expressions “consisting of,” and“consisting, essentially of”. The use of singular identifiers such as“the,” “a,” or “an” is not intended to be limiting solely to the use ofa single component, but may include multiple components. For example,unless stated otherwise the expression “a compound” has the meaning of“one or more compound(s)”. As used herein, “plurality” is understood tomean more than one. For example, a plurality refers to at least two,three, four, five, or more. Unless specifically stated or obvious fromcontext, as used herein, the term “or” is understood to be inclusive.The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements. Ranges are usedherein as a shorthand for describing each and every value that is withinthe range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).Any value within the range can be selected as the terminus of the range.Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. If notindicated differently, “about” can be understood as within 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the statedvalue. Unless otherwise clear from context, all numerical valuesprovided herein can be modified by the term “about”.

As used herein the term “room temperature”, unless specified otherwise,means the ambient temperature of a typical laboratory, which is usuallyabout 18° C. to 25° C. In a specific embodiment, room temperature is atemperature selected from the group consisting of 18° C., 19° C., 20°C., 21° C., 22° C., 23° C., 24° C., and 25° C. As used herein, a“purified” or “isolated” compound means the compound has been separatedfrom the mixture in which it was formed. With respect to cells, a“purified” or “isolated” cell denotes one or more cells or a group ofcells that were enriched or separated from a mixture of cells. By way ofnon-limiting example, nucleated cells can be isolated from the mixtureof cells in an ex vivo sample of whole blood by lysing the erythrocytespresent in the sample and separating by centrifugation intact nucleatedcells, discarding the supernatant and re-suspending the pelleted cellsin a physiological buffer or in a cell culture medium.

Disclosed herein is a “composition”, the term being understood assignifying the product of combining and mixing a plurality ofingredients. A “liquid” composition is a composition which is present inthe liquid state of aggregation. Specifically, the liquid composition asdisclosed herein is an aqueous composition, a solution with water assolvent and a plurality of water-soluble compounds dissolved therein. An“analyte” in general is a substance, specifically a cellular component,which is the target of an analytical process such as, but not limitedto, a process for qualitative or quantitative detection of thesubstance. A specific analyte for the purpose of the present disclosureis selected from DNA, RNA and protein, the latter comprisingoligopeptides, polypeptides, and posttranslational modified derivativesthereof.

A large number of processes are known for purifying and analyzingnucleic acids. In the analytical field, specific focus is on qualitativeand/or quantitative detection of specific DNA or RNA sequences. Usingthe polymerase chain reaction (PCR) and specific primers hybridizing tothe flanks of a DNA sequence of interest, said sequence can beamplified, and the amplified sequence can be detected. Such detectionusually is performed as a hybridization reaction with a labeled probe.So-called real-time PCR allows this detection step to take place whilethe target sequence is being amplified. For the specific detection of aRNA sequence, RNA needs to be reverse-translated to form cDNA; the cDNAreflecting the target RNA sequence can then be amplified and detectedusing a PCR-based DNA amplification technique.

Specific detection of a target protein is typically achieved making useof specific binders capable of binding to the target. Thus, immunoassaydetection methods are a non limiting example of this approach.

The present disclosure is specifically directed to stabilizing ananalyte selected from DNA, RNA and protein in intact nucleated cells ofa whole-blood sample ex-vivo for a period of up to 72 hours (0 to 72 h),specifically 48 to 72 hours, the period counted from the time point ofsampling the whole blood and contacting the whole blood with thestabilizing composition as disclosed herein. Advantageously, the timebetween blood sampling and contacting the blood with the stabilizingcomposition is less than 30 seconds.

The term “stabilizing” includes the specific meaning of keeping intactthe cell membranes of nucleated cells of the blood sample, andpreserving and maintaining in the nucleated cells the cellular proteinsand the cellular DNA as well as the cellular RNA and particularly thecomposition of cellular mRNA reflecting the gene expression status atthe time of sampling; in addition, “stabilizing” encompasses providingan environment in which nucleated cells have a substantially reducedtendency to undergo necrosis or apoptosis, and in which the pattern ofexpressed proteins remains largely unchanged. The term “stabilizing”further includes supporting of vital cell functions, avoidingintoxication of cells and cell death, maintaining the nucleated cells ina resting but viable state.

Unless explicitly mentioned otherwise, the expression “citrate/citricacid” refers to a mixture of trisodium citrate and citric acid.Reference to Blood with EDTA as the sole anticoagulant means whole bloodhaving an EDTA concentration of about 1.8 mg per ml of blood.

A first aspect as disclosed herein is a liquid composition forstabilizing an analyte in intact nucleated cells of a whole-blood sampleex-vivo, the composition being an aqueous solution, the solutioncomprising an anticoagulant, a phosphate salt, a cell-metabolizablesugar, adenine, and an antioxidant, wherein the antioxidant comprises amitochondria-targeted antioxidant, preferably a mitochondria-targetedantioxidant selected from the group consisting of SkQ1, MitoQ, SS-31,and a mixture thereof. Specifically, the analyte is selected from DNA,RNA and protein of intact nucleated cells of the whole-blood sample.

The term “whole blood” in all aspects and embodiments disclosed hereindenotes blood that was directly collected from a vertebrate,specifically from a mammal, more specifically from a primate, even morespecifically from a human, without any further treatment other than theblood drawing process. It is understood that the blood drawing processitself is not part of any of the procedures, uses and/or methodsaccording to the present disclosure. Rather, the present disclosurerelates to procedures, uses and methods based on the provision of asample of whole blood “ex-vivo”, that is to say whole blood outside ofand separate from the organism from which it was drawn. The whole bloodex vivo is provided as a “sample”, being understood as a measured amountof whole blood.

Additives that inhibit blood and/or plasma from clotting are importantfor ensuring that the cells to be analyzed are not negatively affectedby blood coagulation prior to the analytical process. Anticoagulationoccurs by binding calcium ions (EDTA, citrate) or by inhibiting thrombinactivity (heparin, hirudin). Therefore, in order to prevent bloodclotting of the ex-vivo whole blood sample, the composition as disclosedin all aspects and embodiments herein comprises an anticoagulant,specifically an anticoagulant selected from the group consisting ofEDTA, citrate, heparin, hirudin, warfarin, and a mixture thereof.Anticoagulant additives which at the same time permit integrity, that isto say the absence of lysis and the viability of nucleated cells areknown to the art. In a specific embodiment in all aspects as disclosedherein, sodium citrate/citric acid is added to the whole blood sample toa final concentration of 11.7 mM and 2.1 mM, respectively.

The phosphate salt in the composition as disclosed in here, either aloneor in combination with EDTA and/or citrate, if present, effects thebuffering of the whole blood sample at a physiological pH. In a specificembodiment of all aspects as disclosed herein, the phosphate salt isNaH₂PO₄, added to the whole blood sample to a final concentration of 2.1mM in an even more specific embodiment. Further additives are adenineand a cell-metabolizable sugar, in order to support cell integrity andviability. In a further specific embodiment of all aspects as disclosedherein, adenine is added to the whole blood sample to a finalconcentration of 0.24 mM.

Notably, is was found by the authors of to present disclosure that thepresence of an effective amount of an antioxidant is essential, in orderto stabilize nucleated cells of a whole blood sample ex-vivo. Theobserved beneficial effects of antioxidants point to reactive oxygenspecies as a possible cause for cell instability in the sample. Reactiveoxygen species are typically generated by NADPH oxidase, xanthineoxidase, the mitochondrial electron-transport chain, and dysfunctionalendothelial nitric oxide synthase. When the capacity of cellularantioxidant defense systems, e.g., superoxide dismutase, catalase,glutathione peroxidase, heme oxygenase, paraoxonase, is exceeded, thisresults in oxidative stress, which can promote cell death. Therefore,means to prevent oxidative stress are of major interest.

Particularly, mitochondria produce substantial amounts of O₂ ⁻ at themitochondrial electron-transport chain complexes I and III. Complex Ireleases O₂ ⁻ into the mitochondrial matrix and is considered the mainproducer of O₂ ⁻ due to reverse electron flow from complex II underlow-ADP conditions. The matrix-localized mitochondrial superoxidedismutase 2 (SOD2) dismutates O₂ ⁻ to H₂O₂, which in turn is reduced towater by glutathione peroxidase or catalase. The importance of SOD2 thuslies in the detoxification of O₂ ⁻ to prevent generation of ONOO⁻(peroxynitrite) and/or oxidative damage of mitochondrialelectron-transport chain proteins and mitochondrial DNA, which mayotherwise compromise mitochondrial function. Complex III also releasesO₂ ⁻ to the mitochondrial intermembrane space, where it is dismutated bySOD1, another isozyme of superoxide dismutase. Mitochondria themselvesare known to be sensitive to reactive oxygen species. Oxidative damagelowers their activity and may even increase their production of reactiveoxygen species. Enhanced levels thereof are known to damage themitochondrial DNA and to stimulate the release of mitochondrialapoptotic factors.

Importantly, the composition in all aspects and embodiments as disclosedin here comprises a mitochondria-targeted antioxidant. An “antioxidant”in a generic sense is a molecule that is capable of inhibiting oxidationor reactions promoted by oxygen, reactive oxygen species, and peroxides.This includes the property of being capable of inhibiting and/orterminating a reaction of a free radical. An antioxidant is typically areducing agent which is capable of taking part in oxidation reactions asan electron donor, thus becoming oxidized itself. An antioxidantaccording to the understanding as disclosed in here acts as a substituteoxidation target competing with cellular components which in the absenceof the antioxidant would be oxidized and thereby compromised byoxidative damage.

A “mitochondria-targeted” antioxidant is an antioxidant which istypically applied extracellularly, which is capable of crossing thecellular membrane and capable of interacting with the mitochondrialmembrane, in order to inhibit certain oxidation processes taking placein the mitochondrial compartment of the cell. Specific examples for suchcell-permeable antioxidants are SkQ1, MitoQ, SS-31, among others. SkQ1,also known as a salt comprising 10-(6′-plastoquinonyl)decyltriphenylphosphonium, is known for its neuroprotective properties,among others. When applied to target cells extracellularly, SkQ1 hasbeen shown to inhibit oxidative stress caused by reactive oxygen speciesin mitochondria. MitoQ is a mixture of mitoquinol (reduced form) andmitoquinone (oxidized form), also known as(10-(2,5-dihydroxy-3,4-dimethoxy-6-methylphenyl)decyl)triphenylphosphonium and(10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)decyl)triphenylphosphonium. MitoQ comprises a ubiquinol moiety which iscovalently attached through an aliphatic carbon chain to a lipophilictriphenylphosphonium cation. The antioxidant reactions ofextracellularly applied MitoQ predominantly occur within phospholipidbilayers. SS-31 (d-Arg-Dmt-Lys-Phe-NH2; Dmt=2′,6′-dimethyltyrosine) is amember of a family of several cell-permeable antioxidant peptides thatreduce intracellular free radicals Like other members in the family,SS-31 peptides are known to target mitochondria and protect mitochondriaagainst mitochondrial permeability transition, swelling, and cytochromec release, and prevent t-butylhydroperoxide-induced apoptosis.

A number of other mitochondria targeted antioxidants are known to theart, particularly from clinical applications in the fields ofcardiovascular and neurodegenerative diseases. In principle, these alsoqualify as compounds which can be advantageously used as alternativestabilizing compounds, in order to practice the teachings of the presentdisclosure.

In a specific embodiment of all aspects as disclosed herein, themitochondria targeted antioxidant is SkQ1. In an even more specificembodiment, SkQ1 is added to the whole blood sample to a finalconcentration of 5-500 nM, advantageously to a final concentration of50-250 nM, particularly about 100 nM. In yet a further specificembodiment of all aspects as disclosed herein, the mitochondria targetedantioxidant is SS-31. In an even more specific embodiment, SS-31 isadded to the whole blood sample to a final concentration of 0.1-50 μM,advantageously to a final concentration of 0.5-10 μM, particularly about1 μM.

As the antioxidants disclosed in here are applied in an aqueoussolution, the antioxidants selected to practice all aspects andembodiments of the present disclosure are advantageously water solubleor solubilized in water, e.g. using a helper substance including, butnot limited to a surfactant, a detergent and an emulsifier.

In a specific embodiment of all aspects as disclosed herein, theantioxidant is a mixture of a first and a second antioxidant, whereinthe first antioxidant is a mitochondria-targeted antioxidant, and thesecond antioxidant is an antioxidant other than a mitochondria-targetedantioxidant. This group encompasses water-soluble molecules with areducing activity, wherein the molecules are capable of undergoingoxidation outside of the mitochondrial compartment. The secondantioxidant, in a further specific embodiment, is an antioxidant otherthan SkQ1, MitoQ, and SS-31. Advantageously, in an even more specificembodiment of all aspects as disclosed herein, the second antioxidant isselected from the group consisting of glutathione, acetylsalicylic acid,N-acetyl-5-aminosalicylic acid, N-acetylcysteine,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and amixture thereof.

Further stabilization of nucleated cells of the whole blood sample isprovided by the presence of a protease inhibitor in the liquidcomposition in all aspects and embodiments as disclosed herein. Aspecific embodiment in this regard includes an inhibitor of a serineprotease. A more specific embodiment in this regard includes aninhibitor of human neutrophil elastase. Particular advantage is providedby specific embodiments of a liquid composition for stabilizing ananalyte in intact nucleated cells of a whole-blood sample ex-vivo, theliquid composition including a protease inhibitor selected fromSivelestat, AEBSF, and a mixture thereof. The compound commercialized as“Sivelestat” or “ONO-5046”, also known asN-{2-[({4-[(2,2-dimethylpropanoyl)oxy]phenyl}sulfonyl)amino]benzoyl}glycineis a selective neutrophil elastase inhibitor. AEBSF is an abbreviationof 4-(2-Aminoethyl)benzenesulfonyl fluoride which a water soluble, andwhich is an irreversible serine protease inhibitor. The hydrochloride iscommercially available as Pefabloc® SC. In a specific embodiment of allaspects as disclosed herein, and with particular advantage, Pefabloc® SCis added to the whole blood sample to result in a final concentration of0.05-1 mM, more specifically 0.1-0.5 mM, and even more specifically0.2-0.3 mM, particularly about 0.25 mM.

Pefabloc® SC is a water-soluble serine protease inhibitor with amolecular weight of 239.5 Daltons, also known to the art as4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF). Thecompound is capable of inhibiting proteases like chymotrypsin,kallikrein, plasmin, thrombin, and trypsin.

In order to stabilize the nucleated cells of the whole blood sample, itwas also found to be of advantage to supplement the sample with acell-metabolizable sugar. In a specific embodiment of all aspects asdisclosed herein, the sugar is selected from glucose and fructose. In aneven more specific embodiment, the glucose is added to the whole bloodsample to a final concentration of 15-25 mM, advantageously to a finalconcentration of 19-21 mM, particularly about 20 mM.

A further aspect as disclosed in here is a kit of parts comprising aliquid composition for stabilizing an analyte in intact nucleated cellsof a whole-blood sample ex-vivo as disclosed in here, the compositionbeing an aqueous solution, the solution comprising an anticoagulant, aphosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant,wherein the antioxidant comprises a mitochondria-targeted antioxidant,the liquid composition being enclosed in a blood drawing container, thekit further comprising packaging material, a label, and a userinstruction sheet.

The liquid composition for stabilizing an analyte in intact nucleatedcells of a whole-blood sample ex-vivo in all aspects and embodiments asdisclosed in here is advantageously contained in a blood drawingcontainer. An example therefor is known to the art as a “vacutainer”. Avacutainer is a blood collection tube provided as a sterile glass orplastic tube with a closure and a vacuum inside the tube facilitatingthe draw of a predetermined volume of liquid such as a whole bloodsample from the vein of a subject. A vacutainer tube according to thepresent disclosure contains a liquid composition for stabilizing ananalyte in intact nucleated cells of a whole-blood sample ex-vivo, inorder to stabilize and preserve the whole blood sample prior toanalytical testing. Tubes are available with or without asafety-engineered closure, with a variety of labeling options andclosure colors as well as a range of draw volumes.

In the blood collection process, the vein is first punctured with ahypodermic needle which is carried in a translucent plastic holder. Theneedle is double ended, the second shorter needle being shrouded forsafety by the holder. When a Vacutainer test tube is pushed down intothe holder, its rubber cap is pierced by the second needle and thepressure difference between the blood volume and the vacuum in the tubeforces blood through the needle and into the tube. The filled tube isthen removed and another can be inserted and filled the same way. It isimportant to remove the tube before withdrawing the needle, as there maystill be some suction left, causing pain upon withdrawal. Once thecollection tube is removed from the needle, the whole blood sampleinside the tube is detached from the body of the subject, hence being anex-vivo whole blood sample. Thus, another specific embodiment of allaspects as disclosed herein is enclosing a mixture of (a) a liquidcomposition for stabilizing an analyte in intact nucleated cells of awhole-blood sample ex-vivo as disclosed herein, and (b) an amount ofwhole blood, i.e. the sample being a measured and/or predeterminedamount of whole blood ex-vivo.

Another aspect disclosed in here is the use of a liquid composition forstabilizing an analyte in intact nucleated cells of a whole-blood sampleex-vivo as disclosed in here, the composition being an aqueous solution,the solution comprising an anticoagulant, a phosphate salt, acell-metabolizable sugar, adenine, and an antioxidant, wherein theantioxidant comprises a mitochondria-targeted antioxidant, the use beingfor the purpose of stabilizing an analyte selected from DNA, RNA andprotein in intact nucleated cells of a whole-blood sample ex-vivo.

Thus, another aspect disclosed in here is a method for stabilizingintact nucleated cells of a whole-blood sample ex-vivo, the methodcomprising the steps of (a) providing the whole blood sample ex-vivo;(b) contacting and mixing the sample of step (a) with a liquidcomposition for stabilizing an analyte in intact nucleated cells of awhole-blood sample ex-vivo as disclosed in here, the composition beingan aqueous solution, the solution comprising an anticoagulant, aphosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant,wherein the antioxidant comprises a mitochondria-targeted antioxidant;(c) incubating the mixture obtained in step (b), thereby stabilizingintact nucleated cells of the ex-vivo whole-blood sample. In a specificembodiment of all aspects as disclosed in here, the whole blood samplecontacted and mixed with the liquid composition as disclosed in here canbe incubated at room temperature for a prolonged time interval, wherebythe nucleated cells present in the whole blood sample are stabilized, aswell as analytes in the cells, particularly DNA, RNA, and protein. In aspecific embodiment, the step of incubating the mixture obtained in step(b) can be performed at room temperature for a time interval selectedfrom the group consisting of 0 h-12 h, 0 h-24 h, 0 h-36 h, 0 h-48 h, 0h-60 h, and 0 h-72 h, or even for longer, depending on the analyte to bedetected thereafter.

In the embodiments of all aspects as disclosed herein, the target cellfor stabilization is a nucleated cell present in the ex-vivo sample ofwhole blood. Specifically, the nucleated cell is a nucleated cell ofhematopoetic lineage or a nucleated cell of non-hematopoetic lineage.Specifically, a nucleated cell of hematopoetic lineage is selected fromthe group of cell types consisting of a megakaryocyte, a thrombocyte, anucleated red blood cell, a mast cell, a myeloblast, a basophil, aneutrophil, an eosinophil, a monocyte, a macrophage, a large granularlymphocyte, a small lymphocyte, a T lymphocyte, a B lymphocyte, a plasmacell, and a dendritic cell, or a precursor of any of the said celltypes. In another specific embodiment, the nucleated cell is a nucleatedcancer cell of hematopoetic lineage.

In a further embodiment of all aspects as disclosed herein, the targetcell for stabilization is a nucleated cell present in the ex-vivo sampleof whole blood, the nucleated cell being capable of undergoing two ormore cell divisions thereby forming daughter cells. In a specificembodiment, the nucleated cell is capable of undergoing cell divisionsnot only in vivo but also ex-vivo in a culture medium. Thus, the presentdisclosure, its aspects and embodiments provide liquid compositions forsupporting and/or maintaining the viability of an intact nucleatedtarget cell of a whole-blood sample ex-vivo, the composition being anaqueous solution, the solution comprising an anticoagulant, a phosphatesalt, a cell-metabolizable sugar, adenine, and an antioxidant, whereinthe antioxidant comprises a mitochondria-targeted antioxidant,preferably a mitochondria-targeted antioxidant selected from the groupconsisting of SkQ1, MitoQ, SS-31, and a mixture thereof, wherein theintact nucleated target cell is capable of undergoing one or more celldivisions. More specifically, the nucleated target cell is capable ofundergoing one or more cell divisions in a culture medium.

In this regard, the skilled person is well aware of different culturemedia which support viability and cell division of mammalian cells,specifically of human cells, more specifically of cancer cells of humanorigin. In a specific embodiment the culture media support cellviability and cell division in the absence of a composition forsupporting and/or maintaining the viability of an intact nucleatedtarget cell in an ex-vivo of whole-blood as disclosed in the presentdocument.

On the one hand, despite being in a resting state the viability of thenucleated target cell is maintained in the ex-vivo whole-blood samplethat is mixed with the stabilizing liquid composition as disclosed. Onthe other hand and remarkably, the nucleated target cell which is in aresting state can be isolated from the stabilized blood sample and beput into culture, whereby once removed from the stabilizers thenucleated cell becomes physiologically active again. In other words,upon removing the nucleated target cell from the whole-blood sample withthe stabilizing composition the resting state is reversed, evenincluding the biological functions necessary for cell division. Thus,the skilled person is now equipped with improved means to isolate anucleated target cell from a sample of whole blood, wherein in aspecific embodiment the target cell is capable of undergoing celldivisions ex vivo, and more specifically wherein the target cell is acancer cell, e.g. but not limited to, a circulating tumor cell.

Yet, in a further embodiment of all aspects as disclosed herein there isprovided a method to isolate and culture a nucleated target cell presentin an ex-vivo sample of whole blood, comprising the steps of (a)providing the ex-vivo whole blood sample; (b) contacting and mixing thesample of step (a) with a liquid composition, the composition being anaqueous solution, the solution comprising an anticoagulant, a phosphatesalt, a cell-metabolizable sugar, adenine, and an antioxidant, whereinthe antioxidant comprises a mitochondria-targeted antioxidant,preferably a mitochondria-targeted antioxidant selected from the groupconsisting of SkQ1, MitoQ, SS-31, and a mixture thereof; (c) incubatingthe mixture obtained in step (b), thereby stabilizing intact nucleatedcells of the ex-vivo whole-blood sample; separating nucleated cells fromthe incubated mixture of step (c) and contacting the separated nucleatedcells with a culture medium; and (d) incubating the nucleated cells inthe culture medium. In specific embodiments the liquid composition isselected from a liquid composition for stabilizing an analyte in intactnucleated cells of a whole-blood sample ex-vivo as disclosed herein inany aspects and embodiments.

A cancer with metastatic tumors spread over the human body is moredifficult to remove or treat than a cancer with a primary tumor. In apatient with metastatic disease, circulating tumor cells (CTCs) can befound in venous blood. These circulating tumor cells are potential seedsfor metastatic cancer growth. To detect these cells is a particulardesire in cancer care. Thus, the teaching, aspects and embodimentsdisclosed herein provide additional means for this purpose.

Accordingly, in yet further embodiments of all aspects as disclosedherein, the target cell for stabilization is a nucleated cancer cellpresent in the ex-vivo sample of whole blood. Specifically, the targetcell for stabilization is a nucleated cell present in the ex-vivo sampleof whole blood, wherein the individual from whom the sample of wholeblood is obtained prior to stabilization (and detection) suffers from acancer. In a specific embodiment, the target cell is a nucleated cell ofnon-hematopoetic lineage, particularly a cancer cell of non-hematopoeticlineage. Particularly, the cancer cell is a circulating tumor cell,specifically a cell shedded from a solid tumor into the circulation, thecirculation including lymph and blood.

In a specific embodiment of all aspects as disclosed herein, thecirculating tumor cell is selected from a solid tumor of cells selectedfrom the group consisting of epithelial, connective tissue, bone,cartilage, muscle, and nerve cells. Circulating tumor cells are specificnucleated cells the present disclosure as presented here aims to makedetectable in whole blood, by stabilizing the cancer cells in the wholeblood over a period of time, preferably at room temperature.

The following examples, figures, and the sequence listing are providedto aid the understanding of the present invention, the true scope ofwhich is set forth in the appended claims. It is understood thatmodifications can be made in the procedures set forth without departingfrom the spirit of the disclosures and teachings as provided herein.

Example 1 Preservation of Cellular Proteins

Suspended cells of MDA-MB-468, a human breast cancer cell line werespiked into blood samples obtained from healthy individuals to result in100,000 spiked cells per ml blood. The samples were processed eitherimmediately or after storage at room temperature for 48 hours. Sample 1contained EDTA as anticoagulant only and no additive, sample 2 to 6 weresupplemented with Sivelestat (10 μg/ml), Pefabloc® SC (1 mM), SkQ1 (100nM), Mito Q (100 nM) and glutathione (3 mM), respectively.

Nucleated cells were enriched by erythrocytes lysis. A 50 μl aliquot ofpeach sample was mixed with 200 μl of lysis buffer (100 mM NH₄Cl, 5 mMHepes, 0.5 mM KHCO₃, 0.1 mM EDTA) and incubated for 10 min at roomtemperature. After centrifugation at 200×g for 15 min, the supernatantwas removed and the pellet was resuspended in 250 μl of lysis buffer.After centrifugation at 200×g for 15 min, the supernatant was removedand the pellet was resuspended in 1,000 μl of PBS, 0.3 mM EDTA. Thecells were sedimented by centrifugation at 200×g for 15 min. The pelletwas resuspended in PBS, 0.3 mM EDTA and the volume of the samplesadjusted to 500 μl.

For each sample a volume of 50 μl of the cell suspension was spotted ona glass slide and air-dried. The slides were fixed in ice cold acetonefor 10 min, washed with PBS twice and immersed in labeling mixturecontaining anti-cytokeratine K5/K8—FITC labeled antibody diluted 1:50 inPBS (cytokeratine=CK). After an incubation for 30 min in the dark thesupernatant was removed. DAPI-containing mounting medium was added, thespots covered with cover slips and after a further incubation for 20 minanalyzed on a Zeiss Axio Observer Microscope. The total number ofnucleated cells and the CK 5/8 positive cells were analyzed with theAssay Builder Software from Zeiss. The result is shown in FIG. 1. Thesamples containing protease inhibitors like Sivelestat (inhibitor ofneutrophilic elastase) and Pefabloc SC (serine protease inhibitor)showed higher signals than the control sample without additive.Particular positive effects were observed with SkQ1 and MitoQ,antioxidants targeted to mitochondria. A further positive effect wasobserved with the antioxidant glutathione.

FIG. 1 illustrates the detection of MDA-MB-468 cells spiked into bloodsamples after storage for 48 hours at room temperature. Depicted is thepercentage of MDA-MB-468 cells detected by immunostaining with Anti-CK5/8 antigens.

Example 2 Preservation of Cellular Proteins

MDA-MB-468 cells were spiked into blood samples obtained from healthyindividuals to result in a concentration of 100,000 spiked cells/mlblood. The samples were processed either immediately or after storage atroom temperature for 48 hours. Sample 1 contained EDTA as soleanticoagulant and no further additive, samples 2 to 4 contained citrate(11.7 mM)/citric acid (2.1 mM), NaH₂PO₄ (2.1 mM), Dextrose (20.1 mM;Dextrose=D-glucose), Adenine (0.24 mM) as stabilizer. Sample 3 wasadditionally supplemented with the mitochondria targeted antioxidantSS-31 (1 μM), sample 4 with glutathione (0.75 mM). Nucleated cells wereenriched by erythrocytes lysis and processed as described in Example 1.

The result is shown in FIG. 2. Further to the results of Example 1,antioxidants also showed their positive effect in citrate anticoagulatedblood. FIG. 2 illustrates the detection of MDA-MB-468 cells spiked intoblood samples after storage for 48 hours at room temperature. Depictedis the percentage of MDA-MB-468 cells detected by immunodetection withfluorescent Anti-CK5/8 antibodies.

Example 3 Preservation of DNA

Blood samples from 3 healthy individuals in EDTA or EDTA mixed withglutatione (325 mM), SKQ1 (100 μM), and Pefabloc®SC (100 mM), alsoreferred to as “Stabilizer 3”, were spiked with Karpas 422 cells infinal concentrations of 200,000 spiked cells per ml blood. The sampleswere stored at ambient temperature. Karpas cells are a B-cellnon-Hodgkin's lymphoma (NHL) cell line bearing both t(14; 18) and t(4;11) chromosomal translocations. The t(14; 18) translocation was selectedas a marker to distinguish the DNA originating from Karpas cells fromthe DNA originating from normal, i.e. t(14; 18) translocation-free bloodcells.

Immediately after sample set up and following an incubation at roomtemperature for 48 hours, nucleated cells were enriched by erythrocytelysis. Aliquots of 350 μl sample were mixed with 1,000 μl of lysisbuffer (80 mM NH₄Cl, 10 mM Hepes buffer, 0.1 mM EDTA) and incubated for10 min at room temperature. After centrifugation at 300×g for 15 min,the supernatant was removed and the pellet was resuspended in 1,000 μlof lysis buffer. After centrifugation at 300×g for 15 min, thesupernatant was removed and the pellet was resuspended in 1,000 μl ofPBS. The cells were sedimented by centrifugation at 300×g for 15 min.The pellet was resuspended in PBS and the DNA isolated with High PureTemplate Preparation Kit (Roche Applied Science, Cat. 11 796 828 001)according to the protocol recommended by the manufacturer. The DNA waseluted with 100 μl elution buffer from the HighPure columns.

Aliquots of 5 μl DNA from each sample were used in 20 μl PCR reactions,in triplicates. Amplification was performed with Lightcycler 480 ProbesMaster (Roche Applied Science Cat. No. 04707494001) in a LightCycler 480instrument with an initial denaturation at 95° C. for 10 min, 50 cycleswith 95° C. for 10 sec, 60° C. for 60 sec. The primer and probesequences used were: t(14/18) forward primer acctgaggagacggtgac (SEQ IDNO:1); t(14/18) reverse primer tggggttttgacctttagaga (SEQ ID NO:2);t(14/18)-FAM detection probe 6FAM-ctctgggtgggtctgtgttgaaaca-BHQ2(sequence is SEQ ID NO:3).

The differences in crossing points (term definition see Example 4)caused by storage of the samples at room temperature for 48 hours areshown in FIG. 3. There was no significant degradation of DNA in bothconditions tested. In the presence of Stabilizer 3 however, reducedsample-to-sample variation was observed, and also a reduction ofintra-assay variation.

Example 4 Preservation of RNA

Blood samples from breast cancer patients containing circulating tumorcells were collected either in citrate (11.7 mM)/citric acid (2.1 mM),NaH₂PO₄ (2.1 mM), Dextrose (20.1 mM), Adenine (0.24 mM) and SkQ1 (100nM), designated as “Stabilizer 4”, or in CellSave (Veridex Cell SavePreservative tube, Cat. No. 7900005). Samples #59, #60, #62, #63, #58were stored for 48 hours at room temperature, samples #55, #56 and #57were stored for 72 hours at room temperature. Nucleated cells wereenriched by erythrocyte lysis. Aliquots of 350 μl blood sample was mixedwith 1,000 μl of lysis buffer (80 mM NH₄Cl, 10 mM Hepes, 0.1 mM EDTA)incubated for 10 min at room temperature, centrifuged for 15 min at300×g, washed with 1,000 μl of lysis buffer, centrifuged at 300×g for 15min. The pellet war dissolved in 200 μl of PBS and RNA isolated fromthese cells with High Pure RNA Isolation Kit from Roche Applied Science,Cat. No. 11828665001. The columns were eluted with 56 μl of elutionbuffer. 2 μl aliquots from the isolated RNAs was reverse transcribedusing the Transcriptor first strand cDNA Synthesis kit (Roche AppliedScience, Cat. No. 04897030001) in reaction volumes of 20 μl. Aliquots of5 μl cDNA were amplified with the LightCycler 480 Probes Master in aLightCycler 480 instrument. The primers and probes that were used aredescribed in Table 1.

TABLE 1 RNA target forward primer reverse primer detection probe E-5′-GGT TAA GCA CAA 5′-CAC CTG ACC CTT 5′-CAC AGT CAC TGA Cadherin CAGCAA CA-3′; GTA CGT-3′; CAC CAA CGA TAA SEQ ID NO: 4 SEQ ID NO: 5 TCC TCCGA-3′ SEQ ID NO: 6§ cMyc 5′-CCC CTG GTG 5′-CTC ATC TTC TTG 5′-ACA CCGCCC ACC CTC CAT GAG GA-3′; TTC CTC CTC AGA-3′; ACC AGC AGC G-3′ SEQ IDNO: 7 SEQ ID NO: 8 SEQ ID NO: 9§ EpCam 5′-GTT TGC GGA 5′-AGG ATT CAC CTT5′-TGA GAA TAA TGT CTG CAC TTC AG-3′; TAA CAT CTT TTT-3′; TAT CAC TATTGA SEQ ID NO: 10 SEQ ID NO: 11 TCT-3′ SEQ ID NO: 12§ muc 1 5′-ATT TCTGAA 5′-TGG CAC ATC ACT 5′-ATT AAG TTC AGG ATG TTT TTG CAG CAC GCT GA-3′;CCA GGA TCT GTG G-3′ ATT TA-3′; SEQ ID NO: 14 SEQ ID NO: 15§ SEQ ID NO:13 cyclin 5′-CCG TCC ATG 5′-GAA GAC CTC CTC 5′-TCT GTT CCT CGC CGG AAGATC-3′ CTC GCA CT-3′ AGA CCT CCA GCA-3′ SEQ ID NO: 16 SEQ ID NO: 17 SEQID NO: 18$ CAV1 5′-ACA GCC CAG GGA 5′-GGA TGG GAA CGG Roche AppliedScience, AAC CTC-3′ TGT AGA GA-3′ Universal ProbeLibrary SEQ ID NO: 19SEQ ID NO: 20 probe: # 42 CK18 5′-TGA TGA CAC CAA 5′-GGC TTG TAG GCCRoche Applied Science, TAT CAC ACG A-3′ TTT TAC TTC C-3′ UniversalProbeLibrary SEQ ID NO: 21 SEQ ID NO: 22 probe: #78, cat. no.04689011001 PPIA Roche Applied Science, Universal ProbeLibrary HumanPPIA Gene Assay, Cat. No.: 05189268001 TBP Roche Applied Science,Universal ProbeLibrary Human TBP Gene Assay, Cat no: 05189268001 beta2mRoche Applied Science, Universal ProbeLibrary Human β2M Gene Assay, Cat.No.: 05189390001 GAPDH Roche Applied Science, Universal ProbeLibraryHuman GAPD Gene Assay, Cat. No.: 05190541001 §includes 5′ FAM label, and3′-BHQ2 label $includes 5′ FAM label, and 3′-TAMRA label

Real-time PCR including RT (=reverse transcription)-PCR use thelinearity of DNA amplification to determine absolute or relative amountsof a known sequence in a sample. By using a fluorescent reporter in thereaction, DNA generation is monitored at each cycle of PCR. When the DNAis in the log linear phase of amplification, the amount of fluorescenceincreases above the background. The amplification cycle during which thefluorescence becomes measurable against background is called thethreshold cycle or “crossing point”.

Results are shown in Tables 2a-2d. In each section (2a-2d) the firstline indicates the sample number and the respective storage time at roomtemperature. The second line specifies the type of stabilizer reagent.RT-PCR was performed in duplicates, indicated as “Test A” and “Test B”.The numbers in the table are the crossing points obtained afterreal-time RT-PCR. “neg” indicates negative results.

TABLE 2a sample # 59 (48 h) sample # 60 (48 h) Stabilizer 4 CellSaveStabilizer 4 CellSave RNA target Test A Test B Test A Test B Test A TestB Test A Test B E_Cadherin 34.17 34.49 neg neg 31.62 31.42 neg neg cMyc28.29 27.92 38.57 neg 28.49 28.12 38.19 37.11 EpCam 38.84 40.82 neg neg36.07 34.97 neg neg muc 1 35.08 35.1 neg neg 34.94 34.65 neg neg cyclin36.32 37.12 neg neg 34.81 34.81 neg neg CAV 37.08 36 neg neg 36.71 36.16neg neg ck18 32.98 33.14 neg neg 31.53 31.57 42.01 neg PPIA 23.2 23.1735.95 35.67 21.34 21.03 31.83 33.14 TBP 31.13 31.1 neg neg 29.91 29.65neg neg beta2m 20.02 19.98 36.73 36.29 19.09 18.8 34.78 34.67 GAPDH23.83 23.74 37.85 36.76 22.54 22.56 35.02 35.12

TABLE 2b sample # 62 (48 h) sample # 63 (48 h) Stabilizer 4 CellSaveStabilizer 4 CellSave RNA target Test A Test B Test A Test B Test A TestB Test A Test B E_Cadherin 33.34 33.24 neg neg 31.89 32.5  neg neg cMyc28.47 28.45 neg 35.96 26.79 26.81 35.93 35.82 EpCam 38.52 38.36 neg neg39.09 37.92 neg neg muc 1 34.56 34.23 neg neg 32.85 32.66 neg neg cyclin35.84 36.2 neg neg 34.02 34.08 40.97 neg CAV 37.28 36.5 neg neg neg negneg neg ck18 33.61 33.63 neg neg 31.71 31.46 42.48 neg PPIA 23.63 23.3535.67 34.84 21.79 21.74 33.54 32.49 TBP 31.84 30.71 neg neg 29.48 29.25neg neg beta2m 20.77 20.54 35.98 35.32 19.93 19.68 35.58 35.19 GAPDH24.71 24.67 38.54 37.51 23.33 23.11 35.94 35.48

TABLE 2c sample # 55 (72 h) sample # 56 (72 h) Stabilizer 4 CellSaveStabilizer 4 CellSave RNA target Test A Test B Test A Test B Test A TestB Test A Test B E_Cadherin 32.61 32.22 neg neg 36.15 35.12 neg neg cMyc29.34 29.26 37.92 37   30.47 30.3 neg neg EpCam 37.75 38.21 neg neg40.55 39.1 neg neg muc 1 34.92 35.19 neg neg 35.93 36.21 neg neg cyclin36.37 36.67 neg neg 37.82 37.2 neg neg CAV neg neg neg neg 38.04 neg negneg ck18 32.16 32.14 45   neg 33.21 33.02 neg 45 PPIA 21.88 21.76 34.4634.35 23 22.96 35.53 34.66 TBP 30.43 30.25 neg neg 31.94 31.84 neg negbeta2m 22.07 21.88 36   35.55 22.13 21.96 37.05 37.3 GAPDH 24.18 23.9837.78 36.55 24.54 24.61 neg 39.92

TABLE 2d sample # 57 (72 h) sample # 58 (48 h) Stabilizer 4 CellSaveStabilizer 4 CellSave RNA target Test A Test B Test A Test B Test A TestB Test A Test B E_Cadherin 35.33 35.44 neg neg 34.2 34.05 neg neg cMyc29.74 29.33 38.05 38.27 27.8 27.67 38.84 38.06 EpCam 38.67 40.03 neg neg40.72 37.68 neg neg muc 1 35.63 35.15 neg neg 34.5 34.79 neg neg cyclin37.04 36.93 neg neg 35.58 35.37 neg neg CAV neg 37.65 neg neg neg 36.87neg neg ck18 33.33 33.29 neg neg 32.58 32.51 neg 42.45 PPIA 23.51 23.3235.02 35.52 22.66 22.58 35.78 35.02 TBP 31.3  30.99 neg 40.82 31.1 30.88neg neg beta2m 22.07 21.87 39.52 neg 20.59 20.3 36.73 37.8  GAPDH 24.6424.21 38.44 neg 23.95 23.89 39.15 38.82

The RNA molecules indicated above are known to be specific for certaintypes of circulating tumor cells. Thus, the RNAs coding for E-Cadherin,EpCam, Muc 1, cyclin, CAV and CK18 surprisingly remained detectable inthe samples preserved with Stabilizer 4. The cMyc RNA which was alsoexpressed in leukocytes was detectable with the stabilizer reagentCellSave as well, but with a strong shift in crossing points. Thisindicated that the respective RNA level was decreased by several ordersof magnitude.

The housekeeping gene RNAs PPIA, TBP, beta2m and GAPDH used as controlswere detectable in all samples. The samples stabilized with CellSavehowever showed very high crossing points and therefore very low RNAlevels.

This example shows that RNA in blood samples stored for 48 hours or 72hours can be preserved in the Stabilizer 4 reagent.

Example 5 Quantification of RNA from Cells Isolated after 72 h SampleStorage

MDA-MB-468 cells were spiked into blood samples obtained from twohealthy individuals at 14.4×10⁶ cells/ml. From each donor four sampleswere prepared. Sample 1 and 2 contained EDTA as anticoagulant only andno additive, sample 3 and 4 were stabilized with citrate (11.7 mM),citric acid (2.1 mM), NaH₂PO₄ (2.1 mM), Dextrose (20.1 mM), Adenine(0.24 mM), Pefabloc® SC (0.25 mM), SS-31 (1 μM) and acetylsalicylic acid(1 mM) as stabilizer (“Stabilizer 5”). Aliquots from these samples wereprocessed immediately after set up, other aliquots were processed afterstorage at room temperature for 72 hours. MDA-MB-468 cells were isolatedfrom blood by capturing with BreastSelectBeads from Adnagen Cat. No.:T1-508 with 1,000 μl aliquots of sample material according to theprotocol recommended by the manufacturer.

The bead captured purified cells were resuspended in 1,000 μl PBS. With200 μl aliquots RNA was isolated using the MagNA Pure LC DNA IsolationKit—Large volume Cat No 03310515001 using the Program “DNA LVBlood_20_200” and an elution volume of 100 μl.

The RNA was transcribed into cDNA using Transcriptor First Strand cDNASynthesis Kit Cat. No: 04897030001 in reactions of 70 μl volume, eachreaction containing 12.5 μl of isolated nucleic acid. PCR was performedwith the with the LightCycler 480 Probes Master in a LightCycler 480instrument in 20 μl reactions containing 5 μl of the cDNA samples.Initial denaturation was for 10 min at 95° C., 50 cycles with 10 secondsdenaturation at 95° C. and annealing/elongation at 58° C. for 1 min.Detection format: Dual color hydrolysis Probe UPL (Universal ProbeLibrary, Roche Diagnostics GmbH Mannheim, Germany) probe as indicatedbelow. Table 3 describes the primer/probes for the RNA/DNA targetsanalysed.

To quantify the changes in expression level of the RNA target tested,the relative quantification method of Livak and Schmittgen was used(Livak K. J. and Schmittgen T. D., Methods 2001, 25(4) 402-408). Inorder to provide a reference for normalization of the RNA valuesobtained, the GCK target was amplified from the DNA present in the totalnucleic acid preparation.

FIG. 4 show a strong decline in RNA levels from cells isolated fromblood stored in EDTA. When the cells were isolated from blood stored inStabilizer 5, very little influence on the RNA was observed. Theexpression pattern was only slightly affected during storage.

TABLE 3 Target Template forward primer reverse primer detection probe E-RNA 5′-GGT TAA GCA 5′-CAC CTG ACC 5′-CAC AGT CAC TGA Cadherin CAA CAGCAA CA- CTT GTA CGT-3′ CAC CAA CGA TAA 3- SEQ ID NO: 24 TCC TCC GA-3′SEQ ID NO: 23 SEQ ID NO: 25§ EGFR RNA 5′-CAG GAC CAA 5′-CAC ATC TCC5′-TGC AGT CGT CAG GCA ACA TGG-3′ ATC ACT TAT CTC CCT GAA CAT AAC SEQ IDNO: 26 CTT-3′ ATC CTT-3′ SEQ ID NO: 27 SEQ ID NO: 28§ cMyc RNA 5′-CCCCTG GTG 5′-CTC ATC TTC 5′-ACA CCG CCC ACC CTC CAT GAG GA- TTG TTC CTCCTC ACC AGC AGC G-3′ 3′ AGA-3′ SEQ ID NO: 31§ SEQ ID NO: 29 SEQ ID NO:30 CK19 RNA 5′-CGG GAC AAG 5′-CGT ACT GAT 5′-ACC AAG TTT GAG ATT CTT GGTGC-3′ TTC CTC CTC ATG- ACG GAA CAG GC-3′ SEQ ID NO: 32 3′ SEQ ID NO: 34§SEQ ID NO: 33 EpCam RNA 5′-GTT TGC GGA 5′-AGG ATT CAC 5′-TGA GAA TAA TGTCTG CAC TTC AG- CTT TAA CAT CTT TAT CAC TAT TGA 3′ TTT-3′ TCT G-3′ SEQID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37§ Muc 1 RNA 5′-ATT TCT GAA 5′-TGGCAC ATC 5′-ATT AAG TTC AGG ATG TTT TTG CAG ACT CAC GCT GA- CCA GGA TCTGTG G- ATT TA-3′ 3′ 3′ SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40§ SBEMRNA 5′-TCT CTG CCC 5′-TTG GGT AAA 5′-TAT CCA GCT ACT AGA ATC CGA C-3′ACT GGA ATG TCT GGT CCT GCT GA-3′ SEQ ID NO: 41 T-3′ SEQ ID NO: 43§ SEQID NO: 42 Cyclin D1 RNA 5′-CCG TCC ATG 5′-GAA GAC CTC 5′-TCT GTT CCT CGCCGG AAG ATC-3′ CTC CTC GCA CT-3′ AGA CCT CCA GCA-3′ SEQ ID NO: 44 SEQ IDNO: 45 SEQ ID NO: 46$ CAV1 RNA 5′-ACA GCC CAG 5′-GGA TGG GAA RocheApplied Science, GGA AAC CTC-3′ CGG TGT AGA GA- Universal ProbeLibrarySEQ ID NO: 47 3′ probe: # 42 SEQ ID NO: 48 CK18 RNA 5′-TGA TGA CAC5′-GGC TTG TAG Roche Applied Science, CAA TAT CAC ACG GCC TTT TAC TTCUniversal ProbeLibrary A-3′ C-3′ probe: SEQ ID NO: 49 SEQ ID NO: 50 #78,cat. no. 04689011001 VEGFR RNA 5′-TTC CTG ACC 5′-GGT CCC TGT 5′-AAG TGGCTA AGG TTG GAG CAT CTC GGA TAC ACT T-3′ GCA TGG AGT TCT A-3′ SEQ ID NO:52 TGG CAT-3′ SEQ ID NO: 51 SEQ ID NO: 53§ PPIA RNA Roche AppliedScience, Universal ProbeLibrary Human PPIA Gene Assay, Cat. No.:05189268001 GAPDH RNA Roche Applied Science, Universal ProbeLibraryHuman GAPD Gene Assay, Cat. No.: 05190541001 PBGD DNA 5′GGC TCT TTC TGT5′-CCA CAC TCT 5′-TTA CCA AGG AGC CCG GC-3′ CCT ATC TTT ACT- TTG AAC ATGCCC SEQ ID NO: 54 3′ TGG AGA-3′ SEQ ID NO: 55 SEQ ID NO: 56§ GCK DNA5′CTT TCC TGT GAG 5′GCA GAG TTC 5′-CAG AAG GCA GAT GCA CGA AGA-3′ CTCTGG GGT-3′ GAG GGG AGG CAC SEQ ID NO: 57 SEQ ID NO: 58 AGG-3′ SEQ ID NO:59§ §includes 5′ FAM label, and 3′-BHQ2 label $includes 5′ FAM label,and 3′-TAMRA label

Example 6 Isolation of Viable Cells after Sample Storage for 48 Hours

MDA-MB-468 cells were spiked into blood samples obtained from healthyindividuals to result in a final concentration of 300,000 spikedcells/ml blood. The samples were processed either immediately or afterstorage at room temperature for 48 hours. Sample 1 contained EDTA asanticoagulant only and no further additive, sample 2 contained citrate(11.7 mM)/citric acid (2.1 mM), NaH₂PO₄ (2.1 mM), Dextrose (20.1 mM),Adenine (0.24 mM), Pefabloc® SC (0.25 mM), SS-31 (1 μM) andacetylsalicylic acid (1 mM) as stabilizer (“Stabilizer 5”, see Example5).

Immediately after sample set up and 48 hours later nucleated cells wereenriched by erythrocyte lysis. Aliquots of 1 ml blood were mixed with 4ml of lysis buffer (80 mM NH₄Cl, 10 mM Hepes buffer, 0.1 mM EDTA) andincubated for 10 min at room temperature. After centrifugation at 200×gfor 15 min, the supernatant was removed and the pellet was resuspendedin 2 ml of PBS/3 mM EDTA. The cells were sedimented by centrifugation at200×g for 15 min. The supernatant was removed and the cell pelletsresuspended in 300 μl of PBS supplemented with 2 mM EDTA and 0.5% BSA.

From these samples the MDA-MB-468 cells were isolated using CD326(EpCAM) Microparticles from Miltenyi (Cat No 130-061-101), MS columnsand the MACS separator system (Milteny Cat. Nos. 130-042-201,130-042-102) making use of the protocol recommended by the manufacturer.

The enriched cells were suspended in 300 μl of RPMI medium.

The viability of the cells was tested with the Trypan blue exclusionmethod. 10 μl of each sample was mixed with 10 μl of Trypan blue(Sigma-Aldrich T8154). The number of MDA-MB-468 cells excluding the dyewas counted in a Neubauer chamber. The yield of viable cells wascalculated using the cell number spiked into the blood samples as 100%.

Table 4 shows the results obtained with blood samples from two donors.Stabilizer 5 can preserve a significant number of cells. When just EDTAis used as a stabilizing agent the cell viability is negativelyaffected, to a surprisingly significant extent.

TABLE 4 Percentage of isolated viable cells Anticoagulant/ after storagefor 48 h, erythrocyte Donor Stabilizer lysis and immunomagneticenrichment #1 EDTA 16 #1 Stabilizer 5 44 #2 EDTA 9 #2 Stabilizer 5 46

Example 7 Cells Isolated from Stabilized Blood are Culturable

MDA-MB-468 cells treated and isolated as described in Example 6 wereseeded into the wells of an E-Plate 96-well device (Roche AppliedScience, Cat. No. 06472451001, Roche Diagnostics GmbH Mannheim, Germany)for the Real-Time Cell Analyzer (RTCA) MP (xCELLigence system). 50 μlaliquots of cells were seeded into wells containing 50 μl of RPMImedium. Duplicates were used for analysis. The E-Plate was incubatedover night at 37° C. in an incubator to allow attachment of MDA-MB-468cells to the bottom of the E-Plate. In order to remove contaminatingblood derived cells the medium was removed and cells attached to theculture plate were washed 3 times with PBS. 150 μl of fresh RPMI mediumwas added per well and the E-Plate transferred to the Real-Time CellAnalyzer. Cell index was measured once per hour for 101 hours.

The result is shown in FIG. 5. Cell growth can be observed with cellsisolated from blood stored for 48 hours in stabilizer described in thisinvention.

A comparison was made with cells stabilized with EDTA, only. As itturned out, the cells isolated from blood stored with just EDTA for 48hours are not culturable anymore.

1. A liquid composition for stabilizing an analyte in intact nucleatedcells of a whole-blood sample ex-vivo, the composition being an aqueoussolution, the solution comprising an anticoagulant, a phosphate salt, acell-metabolizable sugar, adenine, and an antioxidant, wherein theantioxidant comprises a mitochondria-targeted antioxidant.
 2. Thecomposition of claim 1 wherein the mitochondria-targeted antioxidant isselected from the group consisting of SkQ1, MitoQ, SS-31, or a mixturethereof.
 3. The composition according to claim 1, wherein thecomposition further comprises a protease inhibitor.
 4. The compositionof claim 1, wherein the antioxidant is a mixture of a first and a secondantioxidant, wherein the first antioxidant is a mitochondria-targetedantioxidant, and the second antioxidant is an antioxidant other than amitochondria-targeted antioxidant.
 5. The composition of claim 4,wherein the second antioxidant is selected from the group consisting ofglutathione, acetylsalicylic acid, N-acetyl-5-aminosalicylic acid,N-acetylcysteine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(Trolox) and a mixture thereof.
 6. The composition according to claim 4,wherein the first mitochondria-targeted antioxidant is selected from thegroup consisting of SkQ1, MitoQ, SS-31, and a mixture thereof.
 7. Thecomposition according to claim 1, wherein the cell-metabolizable sugaris selected from the group consisting of glucose and fructose.
 8. Thecomposition according to claim 1, the composition being enclosed in ablood drawing container.
 9. The composition according to claim 1,further comprising an amount of whole blood.
 10. The compositionaccording to claim 9, wherein the composition comprises one or moremitochondria-targeted antioxidant(s) with an aggregate concentration of10 nM to 200 μM.
 11. The composition according to claim 10, wherein thecomposition comprises a concentration of a mitochondria-targetedantioxidant selected from the group consisting of 50 nM-250 nM.
 12. Thecomposition according to claim 11, wherein the composition comprises11.1 mM-12.3 mM trisodium citrate, 2 mM-2.2 mM citric acid, 2 mM-2.2 mMNaH₂PO₄, 19.1 mM-21.1 mM glucose, 0.23 mM-0.25 mM adenine, 0.24 mM-0.26mM AEBSF, 990 nM-1.1 μM SS-31 and acetylsalicylic acid 990 μM-1.1 mM.13. A kit of parts comprising a composition according to claim 8, thekit further comprising packaging material, a label, and a userinstruction sheet.
 14. A method for stabilizing intact nucleated cellsof a whole-blood sample ex-vivo, the method comprising the steps of (a)providing the whole blood sample ex-vivo; (b) contacting and mixing thesample of step (a) with a composition according to claim 1; (c)incubating the mixture obtained in step (b), thereby stabilizing intactnucleated cells of the ex-vivo whole-blood sample.
 15. The methodaccording to claim 14, wherein the mixture obtained in step (b) is acomposition according to claim
 10. 16. The method according to claim 14,wherein step (c) is performed at room temperature for a time intervalselected from the group consisting of 0 h-12 h, 0 h-24 h, 0 h-36 h, 0h-48 h, 0 h-60 h, and 0 h-72 h.
 17. The method of claim 14, the methodfurther comprising the steps of (d) separating intact nucleated cellsfrom the incubated mixture obtained in step (c); and (e) lysing theseparated nucleated cells and detecting an analyte of nucleated cells inthe lysate, wherein the analyte is selected from the group consisting ofDNA, RNA, and protein.
 18. The method according to claim 17, wherein inthe mixture of step (b) the mitochondria-targeted antioxidant is SS-31at a concentration of 500 nM-10 μM.
 19. The method according to claim18, wherein the mixture of step (b) further comprises acetylsalicylicacid at a concentration of 200 nM-10 mM, and AEBSF at a concentration of50 nM-1 mM.
 20. The method according to claim 14, the method furthercomprising the steps of (d) separating intact nucleated cells from theincubated mixture obtained in step (c); (e) contacting the separatednucleated cells of step (d) with a culture medium, and culturing viablecells.